SK287682B6 - Antithrombotic compound, method for producing thereof, pharmaceutical compositions containing the same and use thereof - Google Patents

Abstract

The present invention relates to compounds of the formula (I), wherein R is independently SO3- or CH3-,the spacer is a flexible spacer of a length of 19 atoms; the charge of the pentasaccharide residue is compensated by positively charged counterions; and the total number of sulfate groups in the pentasaccharide residue is 4, 5 or 6; or a pharmaceutically acceptable salt or a solvate thereof, a method for the production thereof. The compounds of the invention have antithrombotic activity and can be used in treating or preventing thrombin-related diseases.

Description

Technical field

The present invention relates to a novel antithrombotic pentasaccharide conjugate, to pharmaceutical compositions containing the compound as an active ingredient, and to a method of using the compound in the manufacture of a medicament.

BACKGROUND OF THE INVENTION

Serine proteases are enzymes that play an important role in the blood coagulation cascade. An important serine protease is factor Xa, which catalyses the conversion of prothrombin to thrombin. Thrombin is the terminal serine protease enzyme in the coagulation cascade. The main function of thrombin is the cleavage of fibrinogen to form fibrin monomers that are crosslinked to form an insoluble gel. In addition, thrombin regulates its own production by activating factors V and VIII in the early stages of the cascade. It also plays an important role at the cellular level where it acts on specific receptors and causes platelet aggregation, activation of endothelial cells and fibroplast proliferation. Thrombin also plays a central regulatory role in homeostasis and thrombus formation.

In the development of synthetic serine protease inhibitors, it has recently been reported that synthetic NAPAP-pentasaccharide conjugate is an antithrombotic agent of dual nature, namely direct antithrombin activity as well as ATIII-mediated anti-Xa activity (ATIII: antithrombin III) (Bioorg. Med Chem Lett 1999, 9 (14), 2013-2018). Although these anti-thrombotic agents may be of interest, HIT will be associated with this compound by cross-reactivity and neutralization by PF4 due to the high sulfate content of the pentasaccharide residue (Thromb. Haem. Suppl. 1997, p363 PD1485).

The compounds of formula (I) have been found to be antithrombotic agents of excellent and advantageous dual nature. The compounds of formula (I) have a pharmacologically interesting half-life allowing for a one-day treatment and are difficult to neutralize with PF4. In addition, the risks of bleeding are low. In principle, the compounds of formula (I) have an attractive combination of pharmacological properties.

SUMMARY OF THE INVENTION

The present invention provides antithrombotic pentasaccharide conjugates of formula (I)

where

R is independently SO ₃ 'or CH ₃ ;

the linker is a flexible linker having a length of 13 to 25 atoms, preferably 16 to 22, and most preferably 19 atoms; the charge of the pentrasaccharide residue is compensated by positively charged counterions; and the total number of sulfate groups in the pentasaccharide residue is 4, 5 or 6; or a pharmaceutically acceptable salt or prodrug or solvate thereof.

The compounds of the invention are useful for treating and preventing thrombin-mediated and thrombin-associated diseases. This includes a number of thrombotic and prothrombotic conditions where a coagulation cascade is activated, including, but not limited to, deep vein thrombosis, pulmonary embolism, thrombof2 lebitis, arterial occlusion due to thrombosis or embolism, or arterial reoclusion, arterial reocclusion restenosis following arterial injuries or invasive cardiological procedures, postoperative venous thrombosis or embolism, acute or chronic atherosclerosis, stroke, myocardial infarction, cancer and metastasis, and neurodegenerative diseases. The compounds of the invention may also be used as anticoagulants in extracorporeal blood circulation, as required in dialysis and surgery.

The compounds of the invention may also be used as in vitro anticoagulants.

The compounds of formula (I) are specifically useful as antithrombotic agents for arterial indications.

Preferred compounds of the invention are those wherein the pentasaccharide residue has the formula:

The chemical nature of the linker is not very important for the antithrombotic activity of the compounds of the invention. Nevertheless, the linker of the compounds of the invention is flexible, meaning that the linker does not contain solid elements such as unsaturated bonds of cyclic structures. Suitable linkers can be readily designed by those skilled in the art. Preferred linkers contain at least one - (CH ₂ CH ₂ O) element. More preferably, the linkers comprise three - (CH ₂ CH ₂ O) elements. The most preferred linker is * - (CH ₂ CH ₂ O) 3- (CH ₂ ) 2 -NH-C (O) - (CH ₂ ) 3 -NH-C (O) -CH ₂ -, the end labeled is attached to the pentasaccharide rest.

Preferred compounds of formula (I) are compounds of formula (Ia) wherein p is 1 to 5, n is 1 to 5, and m is 1 to 2. Most preferred is a compound of formula (Ia) wherein p is 3, n is 3 and m is 1.

A positively charged counterion is H ⁺ , Na ⁺ , K ⁺ , Ca ²⁺ , and the like. Preferably, the compounds of formula (I) are in the form of their sodium salts.

The term "prodrug" means a compound of the invention wherein the amino group of the amidino group is protected, for example, by a hydroxy or (C _1-6 ) alkoxycarbonyl group.

Solvates of the invention include hydrates.

The compounds of the invention, which may exist in the form of the free base, may be isolated from the reaction mixture in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts may be obtained by treating the free base of formula (I) with an organic or inorganic acid such as hydrochloric, hydrobromic, hydroiodic, sulfuric, phosphoric, acetic, propionic, glyic, maleic, malonic, matsulfonic acid, fumaric acid, succinic acid, tartaric acid, citric acid, benzoic acid, ascorbic acid and the like.

The compounds of the invention have a chiral carbon atom and can therefore be obtained as a pure enantiomer, or as a mixture of enantiomers, or as a mixture containing diastereomers. Methods for obtaining pure enantiomers are well known to those skilled in the art, for example by crystallization of salts obtained from optically active acids and a racemic mixture, or by chromatography using chiral columns. For diastereomers, straight-phase or reverse-phase columns may be used.

The compounds of the invention can be prepared by first activating the carboxyl group of the NAPAP analog of formula (II) and then adding the amino group-containing penta-saccharide linker (formula (III)), optionally followed by deprotection of the amidine group.

The carboxyl group in the compounds of formula (II) may be activated as a mixed anhydride or more preferably as an activated ester, such as an ester of A-hydroxysuccinimide, pentafluorophenol or 1-hydroxybenzotriazole. In the coupling step, the benzamidine group in the compound of formula (II) may be unprotected (R 1 = R = H) or may be protected using a carbamate group, preferably allyloxycarbonyl (R ¹ and / or R 2 is H ₂ C = CH-CH- C (O) O) or benzylcarbonyl (R 1 and / or R is PhCH ₂ C (O) O). Allyloxycarbonyl and benzyloxycarbonyl protecting groups may be removed under relatively mild conditions. The allyloxycarbonyl group may be removed using Pd in the presence of a weak nucleophile such as morpholine or a malonic ester. The benzyloxycarbonyl group may be removed under conditions such as hydrogen / Pd (C). Alternatively, synthetic benzamidine precursors such as N-alkoxybenzamidine or N-benzyloxybenzamidine (R '= H, R = alkoxy or benzyloxy) may be used. These synthetic precursors can be transformed into benzamidine using reducing conditions such as hydrogenation (e.g., Fujii, T et al. Chem. Pharm. Bull, 39, 301, 1991 and Fujii, T et al. Chem. Pharm. Bull, 42, 1231, 1994) .

A preferred benzamidine precursor is 1,2,4-oxadiazolin-5-one (-R'-R- = -C (O) O-). This precursor can be converted to benzamidine by hydrogenation (Bolton, R. E. et al., Tetrahedron Letters, Vol. 36, No. 25, 1995, pp. 4471-4474).

The compounds of formula (II) may be prepared by a variety of methods using methods known in the art. A process for the preparation of compounds of formula (II) wherein R 1 = R = H; n is 3 and m is 1 is described in EP 0513543. Compounds of formula (II) in which the amidine is protected, for example an allyloxycarbonyl or benzyloxycarbonyl group, may be prepared from compounds of formula (IV) wherein the amidine is protected by allyloxycarbonyl or benzyl an oxycarbonyl group using methods commonly known in the art for coupling peptide fragments. The carbamates of formula (IV) can be prepared, for example, from the corresponding amidine (formula (IV), R '= R = H) as described in the literature, for example, Weller, T et al., J. Med. Chem. 39, 3119, 1996).

The N-alkoxybenzamidine and N-benzyloxybenzamidine compounds of formula (II) can be prepared from compounds of formula (V) (described in EP 0513543) by reaction of these cyano compounds with Oalkyl-hydroxylamine or O-benzyl-hydroxylamine, followed by removal of the tert-butyl ester. using acidic conditions. Alternatively, the N-alkoxybenzamidine and N -benzyloxybenzamidine compounds of formula (II) can be prepared by first removing the tert-butyl ester from the compound of formula (V) using acidic conditions to obtain a compound of formula (VI) and subsequently reacting the cyano compound with O- alkyl-hydroxylamine or O-benzyl-hydroxylamine.

Compounds of formula (II) wherein -R'-R- = -C (O) O- (1,2,4-oxadiazolin-5-one group) can be prepared from compounds of formula (IV) wherein -R '-R- = -C (O) O-, using methods known in the art for coupling peptide fragments.

Synthesis of amino-oligosaccharide linker residues of formula (III) may be accomplished, for example, using the methods described in EP 0649854. The saccharide residue of the compounds of the invention may be prepared by methods known in the art, for example from WO 99/25720.

The peptide coupling, a procedural step in the described process for preparing the compounds of the invention, can be carried out by methods known in the art for coupling - or condensing - peptide fragments, such as the azide method, mixed anhydride method, activated ester method, or preferably carboimide. a method, in particular by adding catalytic and racemization suppressing compounds such as N-hydroxysuccinimide and N-hydroxybenzotriazole. An overview is in The Peptides, Analysis, Synthesis, Biology, Vol. 3, E. Gross and J. Meienhofer, eds. (Academic Press, New York, 1981) and Bodanszky, M .; Principles of peptide synthesis, Springer-Verlag, 1984.

The amine functional groups present in the compounds may be protected during synthesis by a V-protecting group, which is a group commonly used in the chemistry of peptides to protect α-amino groups such as a tert-butyloxycarbonyl (Boe) group, benzyloxycarbonyl (Z) group, 9-fluorenylmethylcarbonyl (Fmoc) group or phthaloyl (Phth) group. Deprotection can be accomplished in a variety of ways, depending on the nature of these protecting groups. Usually, deprotection is carried out under acidic conditions and in the presence of a radical acceptor. An overview of amine protecting groups and methods for their removal is given in The Peptides, Analysis, Synthesis, Biology, Vol. 3, and further as described by Greene, T. W. and Wuts, P. G. M. in Protective groups in organic synthesis, John Wiley & 1,991th

The compounds of the invention may be administered enterally or parenterally. The precise dose and dosage regimen of these compounds and their pharmaceutical compositions will necessarily depend upon the need of the individual to whom the drug is administered, the degree of disability or need, and the judgment of the attending physician. In general, parenteral administration requires lower doses than other modes of administration that are more dependent on absorption. However, the daily dose for humans is preferably 0.001 to 100 mg per kg body weight, more preferably 0.01 to 10 mg per kg body weight.

The medicament prepared with the compounds of the invention may also be used as an adjuvant medicament in acute anticoagulant therapy. In such a case, the drug is administered with other compounds useful for the treatment of such disease states.

When compounds are mixed with pharmaceutically acceptable excipients, for example, as described in standard reference, Gennaro et al., Remington's Pharmaceutical Sciences, (18th ed., Mack Publishing Company, 1990, see in particular section 8: Pharmaceutical Preparations and Their Manufacture) compressed into a solid dosage form, such as pills, tablets, or can be made into capsules or suppositories. By means of pharmaceutically acceptable liquids, the compounds can also be used in the form of a solution, suspension, emulsion, for example, for use as an injectable preparation, or as a spray, for example, as a nasal spray.

For the preparation of unit dosage forms, for example tablets, it is contemplated to use conventional additives such as fillers, colorants, polymeric binders and the like. In general, any pharmaceutically acceptable additive that does not interfere with the function of the active compound may be used.

Suitable carriers with which the pharmaceutical compositions can be administered include lactose, starch, cellulose derivatives and the like, or mixtures thereof, used in suitable amounts.

The invention is further illustrated by the following examples.

DETAILED DESCRIPTION OF THE INVENTION

Example 1 Abbreviations used:

Ac = acetyl

Bn = benzyl

DBU = 1,8-diazabicyclo [5.4.0] undec-7-ene

DCC = dicyclohexylcarbodiimide

DMF = N, N-dimethylformamide

Su = succinimidyl

Me = methyl

TBTU = 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate

TEA = triethylamine

TFA = trifluoroacetic acid

Z = benzyloxycarbonyl

The compound numbers correspond to the compounds in Schemes 1-7.

Scheme 1

I-OAc i-o

⁵

Scheme 1 - continued

Scheme 2 - continued

ÔMe OMe

Scheme 3

- 14 R '= HR- = H ^ - ** • 15 R' = Z, R ³ = HI- »- 16 R '= Z, R ^j = SO ₃ -

Scheme 4

Scheme 5

Scheme 6

Scheme 7

Ί ”” ί

RN

Compound 3

To a stirred solution of compound 1 (53.6 g, 143.6 mmol) (R. Roy; WKC Park; Q. Wu; SN. Wang, Tetrahedron Lett., 1995, 36 (25), 4377-4380) and compound 2 (27.9 g, 89.3 mmol) (SJ Danishefsky; MP DeNinno; GB Philips; RE Zelle, Tetrahedron, EN, 1986, 42, 11, 2809-2819) in DMF was added sodium hydride (7.7 g 60 % dispersion, 192.2 mmol) at 50 ° C. After 1 hour, the reaction mixture was heated to 120 ° C. After stirring for 5 minutes, the reaction mixture was cooled to 40 ° C and diluted with water and extracted three times with dichloromethane. The combined organic layers were washed with water and concentrated in vacuo to give crude product 3 (54 g). TLC: Rf = 0.23, ether 100%.

Compound 4

To a stirred solution of compound 3 (89.3 mmol) in 800 mL of dry toluene and 800 mL of acetic anhydride was added dropwise a cold solution of 361.5 mL of sulfuric acid in acetic anhydride (16.5 mL of concentrated sulfuric acid and 345.0 mL of acetic anhydride) at temperature - 30 ° C. After 2 hours, the reaction mixture was quenched with 240 mL of TEA and stirred at room temperature. To the solution was added aqueous sodium bicarbonate (5%), and the aqueous layer was extracted three times with ethyl acetate. The combined organic layers were washed twice with water and concentrated in vacuo. This procedure was repeated to give crude product 4 (53 g). TLC: Rf = 0.29, ether 100%.

Compound 5

To a stirred solution of compound 4 (89.3 mmol) and ethanethiol (11.1 mL, 150.3 mmol) in 370 mL dry toluene was added dropwise a solution of BF _3- etherate in toluene (23.9 mL) at 0 ° C. BF _3- etherate and 190 ml toluene). After stirring at room temperature for 16 hours, the reaction mixture was quenched with TEA and aqueous sodium bicarbonate solution and extracted three times with ethyl acetate. The combined organic layers were washed with water and concentrated in vacuo. The crude product was purified by column chromatography (toluene / ethyl acetate = 1/1 to 0/1, v / v) to give compound 5 (21.4 g). TLC: Rf = 0.31, toluene / ethyl acetate = 4/6, v / v.

Compound 7

A solution of donor 5 (15.0 g, 30.3 mmol) and acceptor 6 (23.0 g, 30.3 mmol) (WO 99/25720) in dry ether / dichloromethane (232 mL, 3/1, v / v) ) was stirred for 30 minutes under a stream of nitrogen in the presence of activated 4Å molecular sieves (7.6 g). Then a solution of 1,3-dibromo-5,5-dimethylhydantoin (5.5 g, 19.1 mmol) and triflic acid (0.49 mL, 5.6) was added dropwise to the reaction mixture at -20 ° C for 75 minutes. mmol) in dioxane / dichloromethane (69.8 mL, 1/1, v / v). After 30 minutes, TEA (5 mL) was added to the reaction mixture, which was stirred for 10 minutes and then filtered. The filtrate was washed with aqueous sodium thiosulfate solution (10%) and aqueous sodium bicarbonate solution (10%), and then concentrated in vacuo. The product was purified by column chromatography (0 to 5% acetone in dichloromethane) to give compound 7 (19.6 g). TLC: Rf = 0.1 ether / heptane = 8/2, v / v.

Compound 8

To a stirred solution of compound 7 (19.5 g, 16.4 mmol) in dry toluene / acetic anhydride (442 mL, 1/1, v / v) was added dropwise a cooled solution of 131.5 mL of sulfuric acid in acetic anhydride (11, 5 ml of concentrated sulfuric acid and 120 ml of acetic anhydride) at -26 ° C. After 75 minutes, TEA (73.5 mL) was added at -20 ° C. Acetic anhydride was reacted by the addition of 330 ml of water in succession, maintaining the temperature between 25 ° C and 30 ° C. After stirring for 16 hours, the mixture was poured into 800 mL of water and extracted twice with toluene. The combined organic layers were washed with water and concentrated in vacuo. The crude product was purified by column chromatography (toluene / ethyl acetate / ethanol = 96/2/2, volume ratio) to give compound 8 as a white foam (13.2 g). TLC: Rf = 0.29, toluene / ethanol = 9/1, v / v).

Compound 9

To a solution of compound 8 (13.2 g, 11.7 mmol) in dry toluene (66 mL) at 32 ° C was added morpholine (4.1 mL, 46.9 mmol). After stirring at 32 ° C for 42 h, the reaction mixture was cooled to room temperature and aqueous hydrochloric acid (17.6 mL, 4N) was added. The mixture was diluted with water and extracted twice with ethyl acetate. The combined organic layers were washed twice with water, dried over sodium sulfate and concentrated in vacuo to give crude compound 9 (12.6 g). TLC: Rf = 0.32, toluene / acetone = 7/3, v / v.

Compound 12

To a solution of compound 9 (12.6 g, 11.6 mmol) in dichloromethane (114 mL) was added trichloroacetonitrile (3.5 mL, 34.9 mmol) and DBU (52.5 μΐ, 0.35 mmol). After stirring at room temperature for 2 hours, to the reaction mixture was added activated 4Å molecular sieves (24 g) and acceptor 11 (8.9 g, 13.0 mmol) (WO 99/25720) in dichloromethane (45 mL). After stirring at room temperature for 30 minutes, the mixture was cooled to -20 ° C and a solution of trimethylsilyl trifluoromethanesulfonate (405 µL, 2.1 mmol) in dichloromethane (100 mL) was added dropwise. After stirring for 30 minutes, sodium bicarbonate was added at -20 ° C and the reaction mixture was filtered. The filtrate was poured into aqueous sodium bicarbonate and extracted three times with dichloromethane. The combined organic layers were washed twice with water and concentrated in vacuo. The product was purified by column chromatography (1: SiO ₂ : 0 to 10% acetone in ether; 2: SiO ₂ toluene / acetone = 85/15 to 80/20, 3: RP-18: water / acetonirtril = 2/8 to 0 (10 volume ratio) to give pure compound 12 (8.9 g). TLC: Rf = 0.37, toluene / acetone = 7/3, v / v.

Compounds 14

A suspension of compound 12 (8.9 g, 5.1 mmol) and 10% Pd / C (8.9 g) in 312 mL of DMF and 45 mL of water was stirred under a continuous stream of hydrogen. After 4.5 hours, the Pd / C catalyst was removed by filtration. The filtrate was concentrated to a volume of 400 mL and treated with 10% Pd / C (1.5 g) under a stream of hydrogen for 5.5 hours. The catalyst was removed by filtration. To the filtrate (900 mL) was added aqueous sodium hydroxide solution (32 mL, 4N). After stirring for 4 hours at room temperature, the mixture was acidified to pH = 6.6 with 1N hydrochloric acid and then concentrated in vacuo. The crude product was desalted on a Sephadex G-25 column which was washed with water. Appropriate fractions were combined and lyophilized to give compound 14 (4.0 g).

Compound 15

Pentasaccharide 14 (700 mg, 0.61 mmol) was dissolved in water (13.2 mL) and DMF (3.3 mL) and treated with N- (benzyloxycarbonyloxy) succinimide (224 mg, 0.90 mmol) and V -ethylmorpholine (233 μΐ, 1.83 mmol). After stirring for 15 minutes, the reaction mixture was directly applied to an RP-18 column which was eluted with water / acetonitrile 10/0 to 7/3. Appropriate fractions were combined and concentrated to a small volume and then applied to a Dowex 50 WX4-H ⁺ ion exchange column in water. The eluate was concentrated in vacuo to give pure compound 15 (482 mg).

Compound 16

To a solution of 15 (471 mg, 0.37 mmol) in DMF (4.7 mL) was added sulfur trioxide pyridine complex (1.1 g, 6.6 mmol) and the mixture was stirred at 30 ° C for 16 h. . The mixture was cooled to room temperature and added dropwise to a cooled 10% sodium bicarbonate solution (16.7 mL, 19.9 mmol) and stirred at room temperature for 1 hour. The mixture was concentrated to a small volume and applied to a Sephadex G-25 column which was eluted with water. Appropriate fractions were combined and concentrated to a small volume, which was then passed through a Dowex Na ⁺ HCRW2 column and eluted with water. The eluate was concentrated and redissolved in 8.3 mL of 0.2 N hydrochloric acid and allowed to stand at 4 ° C for 16 hours. The reaction mixture was neutralized with 8 mL of 0.2N sodium hydroxide and desalted on a Sephadex G-25 column, which was eluted with water. The appropriate fractions were combined and concentrated in vacuo to give compound 16 (840 mg).

Compound 17

A suspension of compound 16 (0.37 mmol) and 10% Pd / C (820 mg) in tert-butanol (85 mL) and water (79 mL) and a few drops of acetic acid was stirred under a continuous stream of hydrogen. After 3 hours, the Pd / C catalyst was removed by filtration and the filtrate was concentrated and lyophilized to give pure compound 17 (675 mg).

4 - [[4 - [[(1R) -1 - [[4- (aminoiminomethyl) phenyl] methyl] -2-oxo-2- (1-piperidinyl) ethyl] amino] -3 - [[(4) benzyl ester hydrochloride -methoxy-2,3,6-trimethylphenyl) sulfonyl] amino] -1,4 (S) -di-oxobutyl] amino] butanoic acid (18)

To a solution of 4 - [[(1 R) -1 - [[4- (aminoiminomethyl) phenyl] methyl] -2-oxo-2- (1-piperidinyl) ethyl] amino] -3 - [[(4-methoxy) hydrochloride solution -2,3,6-trimethylphenyl) sulfonyl] amino] -4-oxo- (3S) -butanoic acid (2.38 g, 3.96 mmol) (Tetrahedron 51, 12047-12068, 1995) and benzyl (4) benzenesulfonate (4). (aminobutyric acid) (1.52 g, 3.96 mmol) (J. Am. Chem. Soc. 105, 5278-528, 1983) in DMF (40 mL) was added under a nitrogen atmosphere.

N, N -diisopropylethylamine (0.689 mL, 3.96 mmol) and tetramethylbenzotriazolyl-uronium tetrafluoroborate (1.91 g, 5.94 mmol). The pH of the reaction mixture was maintained at 6 using N, N -diisopropylethylamine. The reaction mixture was stirred at room temperature for 4 days, concentrated, dissolved in ethyl acetate, washed with 5% sodium carbonate

0.1N hydrochloric acid, dried over magnesium sulfate and concentrated. The residue was dissolved in dry ethanol (5 mL), precipitated with dry diisopropyl ether and filtered to give 2.47 g of the title compound 18. R _f = 0.8, ethyl acetate / pyridine / acetic acid / water = 88/31/18 / 7, volume ratio; Mass (ESI ⁺ ): 777.4 [M + H] ⁺ .

4 - [[4 - [[(1 R) -1 - [[4- (aminoiminomethyl) phenyl] methyl] -2-oxo-2- (1-piperidinyl) ethyl] amino] -3 - [[( 4-Methoxy-2,3,6-trimethyl-phenyl) -sulfonyl] -amino] -1,4 (S) -dioxo-butyl] -amino] -butanoic acid (19)

A suspension of compound 18 (2.42 g, 3.11 mmol) and 10% Pd / C (400 mg) in methanol / water (40 mL, 3/1, v / v) was stirred under a continuous stream of hydrogen. After 8 hours, the reaction mixture was filtered, the filtrate was concentrated and re-evaporated three times with methanol / toluene (1/10, v / v). The residue was dissolved in dry ethanol (5 mL), precipitated with dry diethyl ether, filtered and dried. The residue was dissolved in water and directly poured onto a preparative HPLC DeltaPak RP-C] ₈ using a gradient elution system of 20% AJ 60% B / 20% Caž20% A / 14% B / 66% C over 60 min at a flow rate of 40 ml / minute (A: 0.5 M phosphate buffer pH 2.1; B: water; C: acetonitrile / water = 6/4). Yield 598 mg.

Rf = 26.4 minutes (3 to 10 minutes: 20 to 43% C + 20% A; 10 to 50 minutes: 43 to 66% C + 20% A), (A: phosphate buffer pH 2.1; B: water: C: acetonitrile / water = 6/4, v / v), analytical HPLC supelcosil LC-18DB; Mass (ESI ⁺ ): 687.2 [M + H] ⁺ , (ESP): 685.2 [MH],

Compound 21 prepared from compound 17 and compound 19

To a solution of compound 19 (40 mg, 58.3 pmol) in DMF (800 µL) was added N-hydroxysuccinimide (9.0 mg,

78.1 pmol), DCC (18.5 mg, 89.7 pmol) and 1-hydroxybenzotriazole (8.8 mg, 65.1pmol). The reaction mixture was stirred at room temperature for 40 hours. The reaction mixture was filtered through dicalite and the dicalite was washed four times with DMF (284 µL). 0.1 M Na ₂ HPO ₄ buffer (1936 µL, pH = 7.5) and pentasaccharide 17 (94.7 mg, 52.6 pmol) were added to the filtrate. After stirring for 30 minutes, the mixture was filtered through dicalite, concentrated and applied to a Sephadex G-50 column eluted with acetonitrile / water (1/1, v / v). Appropriate fractions were combined, concentrated, and desalted twice by Sephadex G-50 column chromatography (water). Appropriate fractions were combined and lyophilized to give conjugate 21 as a white solid (95.8 mg). Mass (ESI ⁺ ) = 2469, HPLC: Rf = 8.3 min (20 to 80% B for 15 min, A = water / acetonitrile 8/2; B = 2M NaCl / acetonitrile 8/2, volume ratio), analytical HPLC MonoQ HR 5

Compound 22

A solution of (R) -A-Boc (4-cyanophenyl) alanine (25.0 g, 86.1 mmol), piperidine (21.3 mL, 215.3 mmol) and TBTU (41.5 g, 129.2 mmol) in dry CH 2 Cl 2 (500 mL) was stirred at room temperature under a stream of nitrogen for 2 hours. The reaction mixture was washed sequentially with 0.2 N hydrochloric acid, water, aqueous sodium bicarbonate (saturated) and water. The organic layer was dried over MgSO ₄ , filtered and concentrated in vacuo. The product was dissolved in hot ethyl acetate (35 mL), precipitated with heptane (190 mL), and filtered to give compound 22 (27.75 g). TLC: _Rf = 0.58, heptane / ethyl acetate = 3/7, volume ratio.

Compound 23

A solution of compound 22 (25.6 g, 71.7 mmol), hydroxylamine.HCl (7.1 g, 101.8 mmol), and triethylamine (16.8 mL, 120.5 mmol) in complete ethanol (307 mL) was added. was stirred at 80 ° C for 4 hours. After cooling the mixture to room temperature, crystals formed. The crystals were filtered off, washed with ethanol and ether and dried in the oven to give compound 23 (24.5 g). TLC: Rf = 0.15, heptane / ethyl acetate = 3/7, v / v.

Compound 24

A solution of compound 23 (24.5 g, 62.7 mmol) and ethyl chloroformate (7.2 mL, 75.3 mmol) in dry pyridine (245 mL) was stirred at 115 ° C for 2 hours. The reaction mixture was cooled to room temperature and poured into water (1250 mL), then extracted three times with ethyl acetate (500 mL). The combined organic extracts were dried over MgSO ₄ , filtered, and concentrated in vacuo to give compound 24 (24.3 g). TLC: R f = 0.42, CH ₂ Cl 2 / MeOH 95/5, volume ratio.

Compound 25

A solution of compound 24 (24.3 g, 58.4 mmol) in dry CH ₂ Cl ₂ (122 mL) and TFA (122 mL) was stirred at room temperature for 2 hours and concentrated in vacuo in the presence of toluene to afford compound 25 (37). 6 g). TLC: R f = 0.35, CH ₂ Cl 2 / MeOH 8/2, volume ratio.

Compound 26

A suspension of H-Asp- (OtBu) -OH (39 g, 206.35 mmol), 4-methoxy-2,3,6-trimethyl-benzenesulfonyl chloride (62 g, 249.3 mmol), diisopropylamine (89 mL, 635 mmol) ) in DMF (950 mL) and water (450 mL) was stirred at 0 ° C for 3 hours. The reaction mixture was poured into ice / water (5 L) and washed twice with diethyl ether, then the mixture was acidified with aqueous hydrochloric acid (4N, 72 mL) and extracted 3 times with ethyl acetate. The combined ethyl acetate layers were dried over MgSO ₄ , filtered, and concentrated in vacuo to give compound 26 (97.7 g). TLC: Rf = 0.67, CT / CH3 / MeOH 8/2, v / v.

Compound 27

A solution of compound 25 (33.5 g), compound 26 (24.7 g), TBTU (36.8 g, 114.6 mmol) and diisopropylamine (27.2 mL, 194.1 mmol) in dry DMF (670 mL) ) was stirred for 2 hours and then concentrated in vacuo. The residue was dissolved in ethyl acetate (750 mL), washed with aqueous sodium bicarbonate solution (5%, 1250 mL) and aqueous hydrochloric acid (0.1N, 1250 mL), dried over MgSO ₄ , filtered and concentrated in vacuo to afford compound 27 ( 33.8 g). TLC: R f = 0.88, CH ₂ Cl 2 / MeOH 8/2, volume ratio.

Compound 28

A solution of compound 27 (33.8 g, 48.3 mmol) in dry CH ₂ Cl ₂ (170 mL) and TFA (170 mL) was stirred at room temperature for 2 hours and concentrated in vacuo in the presence of toluene to afford compound 28 (32). , 3 g). TLC: _Rf = 0.73, _{CH2 Cl2} / -MeOH 8/2, volume ratio.

Compound 29

A solution of compound 28 (32.3 g), H-GABA-OtBu.HCl (9.5 g, 48.4 mmol), TBTU (29.0 g, 90.5 mmol) and diisopropylamine (25.2 mL, 179 , 8 mmol) in dry DMF (622 mL) was stirred at room temperature for 2 hours and concentrated in vacuo. The residue was dissolved in ethyl acetate (840 mL), washed with aqueous sodium bicarbonate solution (5%, 1400 mL) and aqueous hydrochloric acid (0.1N, 1400 mL), then dried over MgSO ₄ , filtered and concentrated in vacuo. The residue was dissolved in ethanol (75 mL) and slowly added to stirred diisopropyl ether (2990 mL) to give compound 29 as off-white crystals (32.0 g). TLC: _Rf = 0.56, CH ₂ Cl ₂ / MeOH 9/1, volume ratio.

Compound 30

A solution of compound 29 (3.0 g, 3.82 mmol) in dry CH ₂ Cl ₂ (15 mL) and TFA (15 mL) was stirred at room temperature for 2 hours and concentrated in vacuo in the presence of toluene. The residue was purified on silica gel using CH ₂ Cl ₂ / MeOH (0% to 6% MeOH) to give pure compound 30 (1.98 g). TLC: _Rf = 0.56, CH ₂ Cl ₂ / MeOH 8/2, volume ratio.

Compound 31

A solution of compound 30 (900 mg, 1.23 mmol), TBTU (396 mg, 1.23 mmol) and diisopropylamine (215 µL, 1.53 mmol) in DMF (45 mL) was stirred at room temperature for 2 hours. Compound 17 (2.0 g, 1.11 mmol) was added and after stirring for 4 hours, the mixture was concentrated in vacuo to give compound 31 (4.17 g).

Compound 21 of Compound 31

A suspension of compound 31 (4.17 g) and 10% Pd / C (2.8 g) in tert-butyl alcohol (28 mL) and water (56 mL) was stirred overnight under a continuous stream of hydrogen. The Pd / C catalyst was removed by filtration and the filtrate was concentrated in vacuo. The residue was dissolved in water and purified on a Q-separose column. Appropriate fractions were combined, concentrated, and desalted by Sephadex G-25 column chromatography (water). Appropriate fractions were combined and lyophilized to give conjugate 21 as a white solid (1.74 g).

Example 2

The biological activities of the compounds of the invention were determined by the following methods.

I. Antithrombin test

Thrombin (factor Ha) is a factor in the coagulation cascade.

The antithrombin activity of the compounds of the invention was determined spectrometrically by measuring the degree of hydrolysis of the chromogenic substrate s-2238 caused by thrombin. This antithrombin activity assay in a buffer system was used to determine the IC _{50 of the} test compound.

Test medium: Tromethamine-NaCl-polyethylene glycol 6000 buffer (TNP)

Control Compound: 12581 (Kabi)

Vehicle: TNP buffer

The solubilization can be carried out with dimethylsulfoxide, methanol, ethanol, acetonitrile or tert-butyl alcohol, which do not have adverse side effects at concentrations up to 2.5% in the final reaction mixture.

process

Reagents *:

Tromethamine-NaCl (TN) buffer; puff composition: tromethamine (Tris) 6.057 g (50 mmol), NaCl 5.844 g (100 mmol), water to 1 L of the PH solution was adjusted to 7.4 at 37 ° C with HCl (10 mmol.l ¹ ). 2. TNP buffer: polyethylene glycol 6000 was dissolved in TN buffer to give a concentration of 3 g / ^l . 3. S-2238 solution: One vial S-2238 (25 mg Chromogenix, Sweden) is dissolved in 20 ml TN buffer to give a concentration of 1.25 mg mL ¹ (2 mM ^1). 4. Thrombin solution: Human thrombin (1000 NIH units / vial, Enzyme Res. Lab. Inc., USA) is dissolved in TNP buffer to give a stock solution of 50 NIH jednotiek.mľ ^first Immediately before use this solution is diluted with TNP buffer to give a concentration of 30.2 NIH jednotiek.mľ ^first

* - All substances used are of analytical grade,

- ultrapure water (Milli-Q quality) was used for aqueous solutions.

Preparation of solutions of test and control compounds

Test and control compounds were dissolved in Milli-Q water to obtain stock concentrations of 10 ^-2 mol / ^l . Each concentration is stepwise diluted with the vehicle to give concentrations of 10 ^3, 10 ^"4 and 10 ⁵ mol l ^first Dilutions, including stock solutions, were used in the assay (final single concentrations in the reaction mixture: 3.10 ⁴ ; ΙΟ ⁴ ; 3.10 '⁵; ΙΟ ⁵ ; 3.10'⁶; 10 '⁶; 3.10 ⁷ and 10' ⁷ mol / ^l ) .

process

At room temperature, 0.075 ml and 0.025 ml of test compound or control compound solution or vehicle, respectively, were pipetted into the wells of the microtiter plate, and these solutions were individually diluted with 0.115 ml and 0.0165 ml TNP buffer. An aliquot of 0.030 mL of S-2238 solution was added to each well and the plate was 10 min. preheated and preincubated by shaking in an incubator (Amersham) at 37 ° C. After preincubation, the addition of 0.030 mL of thrombin solution to each well initiated hydrolysis of S-2238. The plate was incubated (shaking for 30 seconds) at 37 ° C. Starting 1 min. after the start of incubation, the absorbance of each sample was measured every 90 minutes at 405 nm. using a kinetic reader for microtiter plates (Twinreader plus, Flow Laboratories).

All data was obtained on a personal computer using a data processing program (Biolise). For each compound concentration (expressed in mol-Γ ^{1 of the} reaction mixture) and for the blank sample, absorbance was applied against reaction time in min.

Evaluation of responses

For each final concentration, the maximum absorbance was calculated from the graph. The IC ₅₀ value (final concentration, expressed in pmol ^-1 , causing 50% inhibition of the maximum absorbance of the empty sample) was calculated using the logit transformation analysis of Hafner et al. (Arzneim.-Forsch./Drug Res. 1977; 27 (11)). ): 1871-3).

Antithrombin activity of Example 1: IC ₅₀ value: 17 nm.

II. Antifactor Xa assay

Activated factor X (Xa) is a factor in the coagulation cascade. The anti-Xa activity of the compounds of the invention was determined spectrophotometrically by measuring the degree of hydrolysis of the chromogenic substrate S-2222 developed by factor Xa. This anti-Xa activity assay in a buffer system was used to determine the IC ₅₀ value of the test compound.

Control compound: pentasaccharide Org 31540

Vehicle: TNP buffer

Solubilization can be carried out with dimethylsulfoxide, methanol, ethanol, acetonitrile or tert-butyl alcohol, which do not have adverse side effects up to 1% (for DMSO) and 2.5% (for other solvents) in the final reaction mixture.

process

Reagents *:

Tromethamine-NaCl (TN) buffer; buffer composition: tromethamine (Tris) 6.11 g (50.4 mmol), NaCl 10.17 g (174 mmol), polyethylene glycol 6000 3 g.l ^-1 , water to 11 L. The pH of the solution was adjusted to 7.4 at 37 C HC1 (10 mmol l ^1). 3. S-2238 solution: one vial of S-2222 (25 mg Chromogenix; Sweden) was dissolved in water to give a concentration of 0.375 mg.ml ^L (0.5 mmol.l ^L ). 4. Xa solution: bovine factor Xa (71 nKat vial ¹ ; Kabi Diagnostica) was dissolved in 10 mL of TNP buffer and then further diluted with TNP buffer to give a concentration of 0.75 nKat (1.5 µm ^-1) . is to be freshly prepared. 5. ATIII solution: human ATIII (Chromogenix) was diluted in water to give a concentration of 1 U.ml ^1, after which the solution was further diluted with 3 volumes of TNP buffer to a concentration of 0.25 U.ml ^first 6th standard solution: a stock solution of 5.7 anti-Xa U.ml ¹ Org 31540 was diluted in TNP buffer to 0.05 U.ml ^first 7 Test samples: Each preparation is dissolved in water and diluted with TNP buffer to a concentration of 0, 05 nmol.mľ ^first from each preparation was made a range of 9 dilutions (the dilution factor of 1.5) ·

Determination of Xa activity

Each test sample (0.05 ml) was pipetted into a well of a microtiter plate at room temperature. AT-ΠΙ solution (0.05 ml) was added to each sample and then the plate was shaken using a Vari-shaker. An aliquot of X _and solution (0.05 mL) was pipetted into each well 10 minutes after the addition of the ΑΤ-III solution and the plates were shaken again. Exactly 2 minutes after the addition of X _and solution, 0.1 ml of S-2222 solution was pipetted into each well and the plate was then shaken again. A 12 channel pipette was used for all additions. The residue of X _and catalyzed the hydrolysis of S-2222, which was measured photometrically step, the incubation periods of 2 and 22 minutes respectively at room temperature. Absorbance of each sample was measured at 405 nm using Reader Microeli16, Model 310C (Organon Teknika, Oss, The Netherlands) and the increase in absorbance (AOD) was calculated. Each test sample was determined in duplicate. 10 samples each included an empty sample (0.05 ml TNP buffer).

Calibration curve

A dilution range was made from an aliquot of the standard calibration sample solution (dilution factor 1.4 for Org 31540 samples). The resulting standard samples (approximately 12 samples) should contain between 0.01 and 0.05 anti-X _and U / ml. Within each series, 0.05 ml of each standard sample was tested at least three times as described in the section "Determination of X _and activity". A calibration curve was obtained by constructing a straight line from AOD diameter (blank sample) - AOD diameter (standard sample) log _ against log anti-X _and AOD diameter (standard sample)

U / ml values were obtained using least squares method.

Evaluation of responses

Mean anti-X _and activity in U / ml were determined for each sample using a calibration curve.

Antifactor X _{and the} activities of Example 1: 1012 U / pmol.

Claims (7)

PATENT CLAIMS A pentasaccharide conjugate of formula (I) (I), wherein R is independently SO ₃ 'or CH ₃ ; a linker is a flexible linker with a length of 19 atoms; the charge of the pentasaccharide residue is compensated by positively charged counterions; and the total number of sulfate groups in the pentasaccharide residue is 4, 5 or 6; or a pharmaceutically acceptable salt thereof, or a corresponding compound wherein the amino group of the amidino group is protected with a hydroxyl or C 1-6 alkoxycarbonyl group, or a solvate thereof. The pentasaccharide conjugate of claim 1, wherein the pentasaccharide residue has the formula The pentasaccharide conjugate of any one of claims 1 and 2, wherein the linker is * - (CH ₂ CH ₂ O) ₃ - (CH ₂ ) 2 -NH-C (O) - (CH ₂ ) ₃ -NH-C (O -CH 2 -, and the end labeled is attached to the pentasaccharide residue. A pentasaccharide conjugate according to claim 1 of the formula A process for preparing a pentasaccharide conjugate of formula (I) according to claim 1, comprising 10 steps: activating the carboxyl group of the NAPAP analog of formula (II); 15 - addition of an amino group-containing pentasaccharide linker residue of formula (III); and converting a benzamidine compound wherein the amino group of the amidino group is protected with a hydroxyl or C _1-6 alkoxycarbonyl group, and forming a NAPAP analog of formula (II) by hydrogenation, characterized in that the benzamidine compound is 1,2,4-oxadiazoline-5- ónová skupina. A pharmaceutical composition comprising a pentasaccharide conjugate according to any one of claims 1 to 4 and pharmaceutically acceptable excipients. A pentasaccharide conjugate according to any one of claims 1 to 4 for use as a medicament. Use of a pentasaccharide derivative according to any one of claims 1 to 4 for the manufacture of a medicament for the treatment or prevention of thrombosis or other thrombin-related diseases.